Many pharmaceuticals largely focus on development of small-molecule drugs. These drugs are so-called due to their relatively small size that enables them to diffuse freely throughout the body to reach their target. These molecules are also capable of slipping across the otherwise impermeable cell membrane largely unhindered. The next generation of protein, DNA or RNA based therapies, however, cannot readily cross the cellular membrane and thus require cellular modification to facilitate delivery. Established methods use chemical or physical means to breach the membrane and deliver the material into the cytoplasm. Proper intracellular delivery is an important step in the research, development and implementation of the next generation of therapeutics.
In the electroporation process to deliver materials to a cell, DNA molecules accumulate and interact with the electropermeabilized plasma membrane during the electric pulse. Afterwards, those DNA aggregates are internalized into the cytoplasm and subsequently lead to gene expression (Golzio, M. et al., Proc. Natl. Acad. Sci. 99, 1292-1297 (2002); Paganin-Gioanni, A. et al. Proc. Natl. Acad. Sci. U.S.A. 108, 10443-7 (2011); Rosazza, C. et al., Mol. Ther. 21, 2217-2226 (2013); Boukany, P. E. et al. Nat. Nanotechnol. 6, 747-54 (2011); Teissie, J. et al., Biochim. Biophys. Acta 1724, 270-80 (2005); Yannush, M. L. et al., Annu. Rev. Biomed. Eng. 16, 295-320 (2014); Geng, T. & Lu, C., Lab Chip 13, 3803-21 (2013)). It is unlikely that DNA plasmids could navigate through the viscous and crowded cytoplasm to reach the nucleus simply by diffusion (Lechardeur, D. et al., Adv. Drug Deliv. Rev. 57, 755-767 (2005); Dowty, M. E. et al., Proc. Natl. Acad. Sci. U.S.A. 92, 4572-4576 (1995)). Some work has shown that the transportation of DNA from plasma membrane to nucleus is an active biological process through cytoskeletal transport such as via microtubule and actin networks (Rosazza, C. et al., Mol. Ther. 21, 2217-2226 (2013)). It has been found that microtubule and actin networks play an important role in DNA transportation within the cytoplasm, and the time-scale of such processes can be hours long depending on the cell type. The unclear mechanism and complex nature of DNA transfer between the plasma membrane and nucleus hinders the enhancement of electroporation performance in hard-to-transfect cells. Moreover, the strong fields used in current electroporation techniques can lead to significant damage or death (Yarmush, M. L. et al., Annu. Rev. Biomed. Eng. 16, 295-320 (2014); Geng, T. & Lu, C., Lab Chip 13, 3803-21 (2013)). Technologies that can directly send payloads into cells and cell organelles are needed.